The chromatin remodeller ATRX facilitates diverse nuclear processes, in a stochastic manner, in both heterochromatin and euchromatin

The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.

For the microarray experiment, sample size was determined based on power calculation using ssize package. A sample size of n=20 per group (control and case) was sufficient to detect 99% of the probes with a fold change of 1.5. We used n=20 for the control group and n=28 for the patient group (knowing that a same ATRX mutation can results in variability in phenotypes between patients). No statistical method was used to predetermine sample size for the other experiments. Experiments were performed in 2 to 6 independent replicates for Bio-CAP-seq, ATAC-seq, Capture-C and ChIP-seq as is common in the field (Valle-García et al.,2016, Godfrey et al., 2019and Bozhilov et al., 2021 at the exception of the erythroblast ChIP-seq samples H3K4me1, H3K4me3 and H3K27me3 which were performed in only one independent replicate due to the prioritisation for the use of limited material. Erythroblast ChIP-seq samples for H3K4me1 and H3K4me3 corroborated published data at the alpha-globin cluster (Bozhilov et al., 2021). scRNA-seq was only performed with 1 independent replicate per group due to sample availability but the analysis was performed on 4,400 cells per sample and the results were in line with the patient phenotype as discussed in the manuscript. The number of replicates is specified in a summary table in supplemental information.
none Microarray data were validated using qRT-PCR experiment on 20/21 candidate genes including 17/18 DEGs. ATRX ChIP-seq in LCLs were performed with two different antibodies showing similar results (only peaks detected by both antibodies were kept in the final peak calling including all the samples). For other experiments, replicates from samples derived from the same individual (unaffected donor or ATR-X case) gave similar results, replicates from samples derived from different unaffected individuals gave similar results and replicates from samples derived from different patients reflect the heterogeneity observed in the phenotypes knowing that even siblings with the same ATRX mutation can show variability in the severity of their phenotype as discussed in the manuscript. This is exemplified in Figure 5 by experiments using erythroblasts derived from two siblings having variable degrees of alpha-thalassaemia (case1 with alpha thalassaemia and case2 which has not been diagnosed with alpha thalassaemia (absence of HbH inclusion)). scRNA could not be replicated due to limited sample material. Erythroblast ChIP-seq samples H3K4me1, H3K4me3 and H3K27me3 were not replicated due to prioritisation of the material available. However, ChIP-seq samples for H3K4me1 and H3K4me3 corroborated published data at the alpha-globin cluster (Bozhilov et al., 2021).
Experiments were not randomized. Experimental groups of cells were selected by genotype or patient phenotype.
Investigators were not blinded during experiments or analysis. Blinding was not relevant to this study as the data presented are objectively obtained by quantification of cellular phenotypes through measurements of such as mRNA levels and chromatin interactions and all samples from compared groups were analyzed using the same pipelines and/or scripts. Experimental groups of cells were selected by genotype. This antobody panel used in flow cytometry was setup using Fluorescence minus one (FMOs) and single stained controls on cultured erythroblasts throughout differentiation. This panel is used routinely to monitor erythroid differentiation in our ex-vivo culture system (Scott et al; 2020).
H3K4me3 (07-473, lot: 2664283, Millipore/Merc) "Anti-trimethyl-Histone H3 (Lys4) Antibody is a rabbit polyclonal antibody for detection of Histone H3 trimethylated at lysine 4. Also known as Anti-H3K4me3, this highly specific and well published antibody has been validated in ChIP, DB, WB, PIA, ChIP-seq." (https:// www.merckmillipore.com/FR/fr/product/Anti-trimethyl-Histone-H3-Lys4-Antibody,MM_NF-07-473#) H3K4me1 (ab195391, lot: GR304893-2, Abcam) Abpromise guarantee covers the use of ab195391 in the tested applications including ChIP. (https://www.abcam.com/ab195391.pdf) H3K27me3 (ab6002, lot: GR275911-6-1, Abcam) Abpromise guarantee covers the use of ab6002 in the tested applications including ChIP (https://www.abcam.com/histone-h3-trimethyl-k27-antibody-mabcam-6002-chip-grade-ab6002.html) CD34+ cells were extracted from patients with ATRX or unaffected donor or from leucocyte reduction filters obtained from the National Health Service Blood and Transplant (NHSBT). These cells were then expanded and differentiated in an ex-vivo culture system (Scott et al 2020) Lymphoblastoid cell lines were derived from ATR-X cases or unaffected donors. During CD34+ differentiation, cells were staged using cytospins and FACS using a panel of 6 fluorophore-conjugated monoclonal antibodies (Scott et al 2020). LCLs derived from ATR-X patients were validated for the presence of ATRX mutation. CD34+ differentiation is a primary culture system, where cells are differentiated for 10 days so there are no mycoplasma issues. LCLs were routinely tested for mycoplasma contamination. Note that full information on the approval of the study protocol must also be provided in the manuscript.

ChIP-seq Data deposition
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Not relevant ndividuals with ATR-X syndrome and their unaffected fathers were recruited for this study for lymphoblastoid studies. For erythroid studies in controls either healthy male volunteers were recruited or anonymous leucocyte reduction filters (obtained from the NHSBT) were used.